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Nfκb- and MAP-Kinase Signaling Contribute to the Activation of Murine Myeloid Dendritic Cells by a Flagellin A:Allergen Fusion Protein

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Nfκb- and MAP-Kinase Signaling Contribute to the Activation of Murine Myeloid Dendritic Cells by a Flagellin A:Allergen Fusion Protein Article NFκB- and MAP-Kinase Signaling Contribute to the Activation of Murine Myeloid Dendritic Cells by a Flagellin A:Allergen Fusion Protein Tobias Moeller, Sonja Wolfheimer, Alexandra Goretzki, Stephan Scheurer and Stefan Schülke * Paul-Ehrlich-Institut, Vice President´s Research Group 1: Molecular Allergology; 63225 Langen (Hesse), Germany; [email protected] (T.M.); [email protected] (S.W.); [email protected] (A.G.); [email protected] (S.S.) * Correspondence: [email protected]; Tel.: +49-6103-77-5209 Received: 12 December 2018; Accepted: 12 April 2019; Published: 15 April 2019 Abstract: Fusion proteins incorporating the TLR5-ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. We recently reported a flagellin:allergen fusion protein containing the TLR5-ligand flagellin A (FlaA) from Listeria monocytogenes and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) to prevent allergic sensitization in an experimental mouse model. This study analyzes the signaling pathways contributing to rFlaA:Betv1-mediated pro- and anti-inflammatory cytokine secretion and cell metabolism in myeloid dendritic cells (mDCs) in vitro. The influence of mammalian target of rapamycin (mTOR)-, NF B-, and MAP kinase (MAPK)-signaling on cytokine secretion and metabolic activity of bone marrow (BM)-derived mDCs stimulated with rFlaA:Betv1 were investigated by pre-treatment with either mTOR- (rapamycin), NF B- (dexamethason, BMS-345541, TPCA-1, triptolide, or BAY-11) or MAPK- (SP600125, U0126, or SB202190) inhibitors, respectively. rFlaA:Betv1-mediated IL-10 secretion as well as activation of mDC metabolism, rather than pro-inflammatory cytokine secretion, were inhibited by rapamycin. Inhibition of NFκB-signaling suppressed rFlaA:Betv1-induced IL-12, while inhibition of MAPK-signaling dose-dependently suppressed rFlaA:Betv1-induced IL-10 as well as pro-inflammatory IL-6 and TNF-α production. Notably, with the exception of a partial JNK-dependency, rFlaA:Betv1-mediated effects on mDC metabolism were mostly NF B- and MAPK-independent. Therefore, MAPK-mediated activation of both NFκB- and mTOR-signaling likely is a key pathway for the production of pro- and anti-inflammatory cytokines by flagellin fusion protein vaccines. Keywords: flagellin; TLR5; Bet v 1; birch allergy; vaccine; fusion protein; metabolism; MAPK; NF B; signalling 1. Introduction The incidence of allergic diseases has steadily increased over the last 70 years, causing significant decreases in quality of life in affected patients and economic problems [1,2]. Besides symptomatic treatment or avoidance of the respective allergens, allergen specific immunotherapy (AIT) with allergen extracts is the only disease altering treatment option available so far. Currently, AIT is not convenient for patients due to a multi-year treatment regimen, only partially efficacious for some allergies, and can be hampered by unwanted side effects [3]. To improve AIT, novel vaccine candidates and accompanying adjuvants that increase efficacy while decreasing unwanted adverse-effects are needed [4]. In line with this, TLR-ligands with an intrinsic ability to induce robust innate immune responses are of special interest for their utilization as adjuvants. While for example the TLR4-ligand lipopolysaccharide (LPS), a very well characterized Cells 2019, 8, 355; doi:10.3390/cells8040355 www.mdpi.com/journal/cells Cells 2019, 8, 355 2 of 20 cell wall component of Gram-negative bacteria, has strong immune activating properties, its usage as an adjuvant is strongly limited due to its inherent toxicity [5]. Moreover, nucleic acid-based TLR-ligands such as CpG (TLR9), R848 (TLR7/8), or Poly I:C (TLR3), are effective immune stimulators but are limited in their clinical efficacy due to problems with both toxicity and stability in vivo [5]. Interestingly, vaccines adjuvanted with the TLR5-ligand flagellin [6], a bacterial motility protein forming the body of the bacterial flagellum, were reported to be safe and well tolerated in clinical trials [7,8]. In this context, flagellin was demonstrated to be an effective mucosal adjuvant mediating protective immune responses [9–11]. When using flagellin as an adjuvant one of the major advantages is its proteinaceous nature allowing for the efficient generation of fusion proteins of flagellin and the antigen of choice by recombinant DNA technology. The generated fusion proteins combine antigen and adjuvant (flagellin) into a single molecule, which results in the efficient targeting of antigens to and simultaneous activation of TLR5+ antigen presenting cells (APCs) [11]. Consequently, fusion proteins combining flagellin with different antigens were investigated for their clinical potential to treat different diseases including, among others: influenza [12–14], poxvirus [15], West-Nile-Virus [16], tetanus [17], and Pseudomonas infection [18]. Such fusion proteins are also investigated as vaccines for the treatment of IgE-mediated type I allergies [19–22]. Kitzmüller and colleagues recently described the enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvant activity of flagellin C:Betv1 fusion proteins in human monocyte-derived DCs and T cells from allergic patients [19]. So far, all studies testing such flagellin-containing fusion proteins demonstrated that these vaccine candidates have superior immune activating potential compared to the mixture of flagellin and antigen [12–16,18,20,23,24], making them very interesting vaccine candidates for future human application. Despite their well-described immune activating potential, the mechanisms by which flagellin fusion proteins modulate immune responses are less well understood. In line with results obtained by Kitzmüller et al. [19], we recently showed a fusion protein consisting of the TLR5-ligand flagellin FlaA from Listeria monocytogenes and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) to display strong immune modulating properties both in vivo and in vitro, characterized by the induction of both pro- and anti-inflammatory cytokine secretion from murine myeloid dendritic cells (mDCs) as well as peripheral blood mononuclear cells (PBMCs) from birch allergic patients [22]. Here, the pronounced secretion of the anti-inflammatory cytokine IL-10 was shown to suppress allergen-specific TH1- and especially TH2-responses [22]. Mechanistically, we showed that the activation of mDC metabolism resulted in a predominant production of energy by a high rate of glycolysis and lactic acid fermentation known as the Warburg effect [25] mediated by an activation of mammalian target of rapamycin (mTOR). Moreover, evidence was provided that induction of anti-inflammatory IL-10 secretion by rFlaA:Betv1, but not pro-inflammatory cytokine secretion (IL-1β, TNF-α, or IL-6 ) in mDCs, was dose-dependently inhibited by rapamycin (a specific inhibitor of the mTOR1-complex) and therefore dependent on mTOR activation [22]. Interestingly, inhibition of rFlaA:Betv1-induced IL-10 secretion by rapamycin correlated with enhanced production of the TH1-promoting cytokine IL-12, suggesting these two cytokines to be inversely regulated [22].Taken together, these results showed that the immune-modulatory cytokine secretion induced by this vaccine candidate was linked to the activation of mDC metabolism [22]. While the engagement of mTOR in the rFlaA:Betv1-induced anti-inflammatory IL-10 secretion was elucidated, the mechanism by which such fusion proteins induce pro-inflammatory cytokine secretion remained unclear. Therefore, the aim of this study was to further investigate if MAP kinase- and NFκB-signaling contribute to rFlaA:Betv1-induced pro- and anti-inflammatory cytokine secretion as well as the activation of mDC metabolism. Mitogen-activated protein (MAP) kinases are ubiquitously expressed protein kinases that either auto-phosphorylate endogenous serine and threonine residues or phosphorylate their substrates [26]. MAP kinases (MAPK) regulate cell proliferation, stress responses, apoptosis, and the induction of immune responses [26]. In mammalian cells, MAP kinases belong to three MAPK pathways: the ERK1/2- (p42/44), the c-JUN N-terminal kinase 1, 2, and 3 (SAP/JNK-1/2/3), and the p-38 Cells 2019, 8, 355 3 of 20 MAPK-pathway [26]. The induction of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-2, but also anti-inflammatory IL-10 and therefore the type of immune responses elicited by pathogens can be triggered by the activation of MAPK-signaling [27,28]. Mammalian nuclear factor “kapa-light-enhancer“ of activated B cells (NFκB) is a family of five inducible transcription factors [29]. Under normal conditions, the NFκB proteins are predominantly kept inactive by cytoplasmic association with inhibitory IκB (inhibitor of kappa B) proteins which inhibit NFκB-DNA-binding activity [30]. Activation of NFκB signaling by either pro-inflammatory cytokines, pathogen-associated molecular pattern (PAMP)-mediated activation of pattern recognition receptors (PRRs) of the innate immune system, T- and B-cell receptor signaling, and ligation of lymphocyte co-receptors [29,31,32] results in degradation of the inhibitory IκB proteins, the release of NFκB dimers, and their translocation into the nucleus where they activate pro-inflammatory gene transcription [30]. The aim of the present study was to further characterize the signaling pathways contributing to the induction of pro- and anti-inflammatory cytokine secretion in mDCs and their effect on the activation of mDC
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